Method for treatment of tumors using nucleic acid ligands to PDGF

ABSTRACT

A method is provided for treating solid tumors comprising administering a composition comprising a PDGF aptamer and a cytotoxic agent. In a preferred embodiment the PDGF aptamer is identified using the SELEX process for the Systematic Evolution of Ligands by Exponential enrichment. A method is also provided for reducing the interstitial fluid pressure (IFP) of a solid tumor comprising administering a PDGF aptamer. Finally, a method is provided for increasing the uptake of cytotoxic agents into a tumor comprising administering a composition comprising a PDGF aptamer and a cytotoxic agent.

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication Ser. No. 60/205,006, filed May 17, 2000. This application isalso a continuation in part of U.S. Pat. No. 5,674,685, issued Oct. 7,1997, U.S. Pat. No. 5,668,264, issued Sep. 16, 1997 and U.S. Pat. No.5,723,594, issued Mar. 3, 1998, each entitled “High Affinity PDGFNucleic Acid Ligands,” and U.S. Pat. No. 6,229,002, issued, May 8, 2001,entitled “Platelet Derived Growth Factor (PDGF) Nucleic Acid LigandComplexes.”

FIELD OF THE INVENTION

[0002] This invention relates generally to a method for increasing theuptake of drugs into tumors by treatment with a nucleic acid ligand toPDGF in combination with a cytotoxic agent. The method used foridentifying nucleic acid ligands to PDGF is called SELEX, an acronym forSystematic Evolution of Ligands by Exponential enrichment. The method ofthe present invention is useful for increasing the therapeuticeffectiveness of cytotoxic agents.

BACKGROUND OF THE INVENTION

[0003] The Systematic Evolution of Ligands by Exponential Enrichment(SELEX) process is a method for the in vitro evolution of nucleic acidmolecules with highly specific binding to target molecules and isdescribed in U.S. patent application Ser. No. 07/536,428, filed Jun. 11,1990, entitled “Systematic Evolution of Ligands by EXponentialEnrichment,” now abandoned, U.S. Pat. No. 5,475,096, entitled “NucleicAcid Ligands,” and U.S. Pat. No. 5,270,163 (see also WO 91/19813),entitled “Methods for Identifying Nucleic Acid Ligands,” each of whichis specifically incorporated herein by reference in its entirety. Eachof these applications, collectively referred to herein as the SELEXPatent Applications, describes a fundamentally novel method for making anucleic acid ligand to any desired target molecule.

[0004] The SELEX process provides a class of products which are referredto as nucleic acid ligands or aptamers, each having a unique sequence,and which has the property of binding specifically to a desired targetcompound or molecule. Each SELEX-identified nucleic acid ligand is aspecific ligand of a given target compound or molecule. The SELEXprocess is based on the unique insight that nucleic acids havesufficient capacity for forming a variety of two- and three-dimensionalstructures and sufficient chemical versatility available within theirmonomers to act as ligands (form specific binding pairs) with virtuallyany chemical compound, whether monomeric or polymeric. Molecules of anysize or composition can serve as targets. The SELEX method applied tothe application of high affinity binding involves selection from amixture of candidate oligonucleotides and step-wise iterations ofbinding, partitioning and amplification, using the same generalselection scheme, to achieve virtually any desired criterion of bindingaffinity and selectivity. Starting from a mixture of nucleic acids,preferably comprising a segment of randomized sequence, the SELEX methodincludes steps of contacting the mixture with the target underconditions favorable for binding, partitioning unbound nucleic acidsfrom those nucleic acids which have bound specifically to targetmolecules, dissociating the nucleic acid-target complexes, amplifyingthe nucleic acids dissociated from the nucleic acid-target complexes toyield a ligand enriched mixture of nucleic acids, then reiterating thesteps of binding, partitioning, dissociating and amplifying through asmany cycles as desired to yield highly specific high affinity nucleicacid ligands to the target molecule.

[0005] It has been recognized by the present inventors that the SELEXmethod demonstrates that nucleic acids as chemical compounds can form awide array of shapes, sizes and configurations, and are capable of a farbroader repertoire of binding and other functions than those displayedby nucleic acids in biological systems.

[0006] The basic SELEX method has been modified to achieve a number ofspecific objectives. For example, U.S. patent application Ser. No.07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat. No.5,707,796, both entitled “Method for Selecting Nucleic Acids on theBasis of Structure,” describe the use of the SELEX process inconjunction with gel electrophoresis to select nucleic acid moleculeswith specific structural characteristics, such as bent DNA. U.S. patentapplication Ser. No. 08/123,935, filed Sep. 17, 1993, entitled“Photoselection of Nucleic Acid Ligands,” now abandoned, U.S. Pat. No.5,763,177 and U.S. Pat. No. 6,011,577, both entitled “SystematicEvolution of Ligands by Exponential Enrichment: Photoselection ofNucleic Acid Ligands and Solution SELEX,” describe a SELEX based methodfor selecting nucleic acid ligands containing photoreactive groupscapable of binding and/or photocrosslinking to and/or photoinactivatinga target molecule. U.S. Pat. No. 5,580,737, entitled “High-AffinityNucleic Acid Ligands That Discriminate Between Theophylline andCaffeine,” describes a method for identifying highly specific nucleicacid ligands able to discriminate between closely related molecules,which can be non-peptidic, termed Counter-SELEX. U.S. Pat. No.5,567,588, entitled “Systematic Evolution of Ligands by EXponentialEnrichment: Solution SELEX,” describes a SELEX-based method whichachieves highly efficient partitioning between oligonucleotides havinghigh and low affinity for a target molecule.

[0007] The SELEX method encompasses the identification of high-affinitynucleic acid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX process-identified nucleic acid ligands containingmodified nucleotides are described in U.S. Pat. No. 5,660,985, entitled“High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,”that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat.No. 5,580,737, supra, describes highly specific nucleic acid ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH₂),2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent applicationSer. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method ofPreparation of Known and Novel 2′ Modified Nucleosides by IntramolecularNucleophilic Displacement,” describes oligonucleotides containingvarious 2′-modified pyrimidines.

[0008] The SELEX method encompasses combining selected oligonucleotideswith other selected oligonucleotides and non-oligonucleotide functionalunits as described in U.S. Pat. No. 5,637,459, entitled “SystematicEvolution of Ligands by EXponential Enrichment: Chimeric SELEX,” andU.S. Pat. No. 5,683,867, entitled “Systematic Evolution of Ligands byEXponential Enrichment: Blended SELEX,” respectively. These applicationsallow the combination of the broad array of shapes and other properties,and the efficient amplification and replication properties, ofoligonucleotides with the desirable properties of other molecules.

[0009] The SELEX method further encompasses combining selected nucleicacid ligands with lipophilic compounds or non-immunogenic, highmolecular weight compounds in a diagnostic or therapeutic complex asdescribed in U.S. Pat. No. 6,011,020, entitled “Nucleic Acid LigandComplexes.” Each of the above described patent applications whichdescribe modifications of the basic SELEX procedure are specificallyincorporated by reference herein in their entirety.

[0010] One approach to increasing the effectiveness of existinganti-cancer drugs for the treatment of solid malignancies is to augmentthe uptake of the drugs into tumors, and thereby obtain increasedtherapeutic concentration without elevating the adverse side-effects.Most solid tumors display an increased interstitial fluid pressure(IFP). The molecular mechanisms causing increased tumor IFP are poorlyunderstood. However, tumor stroma involvement in IFP control has beendemonstrated. (Gullino et al. (1964) Cancer Res. 24:780-797; Philips etal. (1990) J. Natl Cancer Inst. 82:1457-1469; Jain (1987) Cancer Res.47:3039-3051). It has been suggested that high tumor IFP prevents drugtransport from the circulation into the tumor interstitium. Thereduction of tumor IFP therefore is a target for efforts to increasetumor drug uptake. (Jain (1996) Science 271:1079-1080). Several agentswhich induce a lowering of IFP, and thereby increase the transcapillarytransport in experimental murine tumors have been identified, includingnicotinamide (Lee et al. (1992) Cancer Res. 52:3237-3240),TNF-α(Kristensen et al. (1996) Br. J. Cancer 74:533-536) anddexamethasone (Kristjansen et al. (1993) Cancer Res. 53:4764-4766).

[0011] Platelet-derived growth factor (PDGF) and the cognate tyrosinekinase receptors are potent mitogens for mesenchymal cells. In additionto its growth promoting effects, PDGF-BB is involved also in theregulation of IFP. After dextran-induced anaphylaxis and lowering of IFPin rat skin, local administration of PDGF-BB results in normalized IFP.PDGF receptors are expressed in the tumor stroma of many common solidtumors, e.g. lung, colon and breast carcinomas. Based on theseobservations the effects of PDGF antagonists on tumor IFP, tumortranscapillary transport and therapeutic effects of cytotoxic drugs wereinvestigated.

SUMMARY OF THE INVENTION

[0012] The present invention includes a method for treating tumors, morespecifically, solid tumors comprising administering to a host atherapeutically effective dose of a composition comprising a PDGFaptamer and a cytotoxic agent. In a preferred embodiment the PDGFaptamer is identified using the SELEX process for the SystematicEvolution of Ligands by EXponential enrichment. The present inventionalso includes a method for reducing the interstitial fluid pressure(IFP) of a tumor, more specifically, a solid tumor comprisingadministering a PDGF nucleic acid ligand. Finally, the present inventionincludes a method for increasing the uptake of cytotoxic agents into atumor comprising administering to a host a composition comprising a PDGFaptamer and a cytotoxic agent. The present invention provides a novelmethod to increase drug uptake and therefore the therapeuticeffectiveness of chemotherapy, by treatment with a PDGF inhibitor inconjunction with the therapeutic agent. As illustrated below, treatmentwith a PDGF aptamer decreases interstitial hypertension in these tumorsthereby increasing the uptake of the therapeutic agent.

BRIEF DESCRIPTION OF THE FIGURES

[0013]FIGS. 1A and B show the molecular description of the PDGF aptamerused in the present study ((SEQ ID NO:1), FIG. 1A) and the aptamer thatwas used as the control. ((SEQ ID NO:2), FIG. 1B).

[0014]FIG. 2 illustrates graphically the reduction in tumor interstitialfluid pressure (IFP) upon treatment of PROb tumors with a PDGF specificaptamer. Average tumor IFP in the animals treated with asequence-scrambled control oligonucleotide (n=5) and PDGF-B specificaptamer (n=7) is depicted. Bars represent S.E.M; * p<0.05

[0015]FIG. 3 depicts the distribution of PDGF β-receptors and PDGF-AB/BBin PROb tumors. Morphological analysis of sections from PROb tumor cellsrevealed three discrete zones in the investigated tumors (FIG. 3A).Towards the exterior a cell rich zone containing proliferative cellsarranged in glandular structures was evident. A zone further towards thecentral part of the tumors contained apoptotic cells and cell debris,whereas the central parts were largely acellular, but containedextracellular matrix deposits. Inflammatory infiltrates were not evidentin the investigated tumors. Immunohistochemical analyses showed thatPDGF-AB/BB was expressed in blood vessels (FIG. 3B) and in the centralparts of tumors by cells morphologically identified as macrophages (FIG.3C). A weak but clearly visible staining of PDGF β-receptors was presentin stromal structures in large parts of the tumors, especially aroundtumor glands (FIG. 3D). PDGF β-receptor staining was completely blockedby addition of a peptide corresponding to the amino acids 981-994 of thehuman/murine PDGF β-receptor at a 10-fold molar excess to the anti-PDGFβ-receptor antibody (FIG. 3E).

[0016]FIG. 4 illustrates graphically the reduction of IFP upon treatmentof SCID-Mice bearing subcutaneous KAT-4 tumors (human anaplastic thyroidcarcinoma with PDGF aptamers.

[0017]FIG. 5 illustrates graphically the increased uptake of thechemotherapeutic agent Taxol upon pretreatment of KAT-4 tumors with PDGFaptamers.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Various terms are used herein to refer to aspects of the presentinvention. To aid in the clarification of the description of thecomponents of this invention, the following definitions are provided.

[0019] As used herein a “nucleic acid ligand” is a non-naturallyoccurring nucleic acid having a desirable action on a target. Nucleicacid ligands are often referred to as “aptamers.” A desirable actionincludes, but is not limited to, binding of the target, catalyticallychanging the target, reacting with the target in a way whichmodifies/alters the target or the functional activity of the target,covalently attaching to the target as in a suicide inhibitor,facilitating the reaction between the target and another molecule. In apreferred embodiment, the action is specific binding affinity for atarget molecule, such target molecule being a three dimensional chemicalstructure other than a polynucleotide that binds to the nucleic acidligand through a mechanism which predominantly depends on Watson/Crickbase pairing or triple helix binding, wherein the nucleic acid liganddoes not have the known physiological function of being bound by thetarget molecule. In the present invention, the target is PDGF or regionsthereof. Nucleic acid ligands include nucleic acids that are identifiedfrom a candidate mixture of nucleic acids, said nucleic acid ligandbeing a ligand of a given target, by the method comprising: a)contacting the candidate mixture with the target, wherein nucleic acidshaving an increased affinity to the target relative to the candidatemixture may be partitioned from the remainder of the candidate mixture;b) partitioning the increased affinity nucleic acids from the remainderof the candidate mixture; and c) amplifying the increased affinitynucleic acids to yield a ligand-enriched mixture of nucleic acids.

[0020] As used herein a “candidate mixture” is a mixture of nucleicacids of differing sequence from which to select a desired ligand. Thesource of a candidate mixture can be from naturally-occurring nucleicacids or fragments thereof, chemically synthesized nucleic acids,enzymatically synthesized nucleic acids or nucleic acids made by acombination of the foregoing techniques. In a preferred embodiment, eachnucleic acid has fixed sequences surrounding a randomized region tofacilitate the amplification process.

[0021] As used herein, “nucleic acid” means either DNA, RNA,single-stranded or double-stranded, and any chemical modificationsthereof. Modifications include, but are not limited to, those whichprovide other chemical groups that incorporate additional charge,polarizability, hydrogen bonding, electrostatic interaction, andfluxionality to the nucleic acid ligand bases or to the nucleic acidligand as a whole. Such modifications include, but are not limited to,2′-position sugar modifications, 5-position pyrimidine modifications,8-position purine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil;backbone modifications, methylations, unusual base-pairing combinationssuch as the isobases isocytidine and isoguanidine and the like.Modifications can also include 3′ and 5′ modifications such as capping.

[0022] “SELEX” methodology involves the combination of selection ofnucleic acid ligands that interact with a target in a desirable manner,for example binding to a protein, with amplification of those selectednucleic acids. Optional iterative cycling of the selection/amplificationsteps allows selection of one or a small number of nucleic acids whichinteract most strongly with the target from a pool which contains a verylarge number of nucleic acids. Cycling of the selection/amplificationprocedure is continued until a selected goal is achieved. In the presentinvention, the SELEX methodology was employed to obtain nucleic acidligands to PDGF. See U.S. Pat. No. 5,674,685, issued Oct. 7, 1997, U.S.Pat. No. 5,668,264, issued Sep. 16, 1997 and U.S. Pat. No. 5,723,594,issued Mar. 3, 1998, each entitled “High Affinity PDGF Nucleic AcidLigands,” and U.S. Pat. No. 6,229,002, issued, May 8, 2001, entitled“Platelet Derived Growth Factor (PDGF) Nucleic Acid Ligand Complexes.”

[0023] The SELEX methodology is described in the SELEX PatentApplications.

[0024] “SELEX target” or “target” means any compound or molecule ofinterest for which a ligand is desired. A target can be a protein,peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor,antigen, antibody, virus, substrate, metabolite, transition stateanalog, cofactor, inhibitor, drug, dye, nutrient, growth factor, etc.without limitation. In this application, the SELEX target was PDGF.

[0025] A “cytotoxic agent” is any substance used to destroy tumor cells.The method of this invention can be used with any systemicallyadministrated cytotoxic agent including, but not limited to, Bleomycin,Cisplatin, and Pt analogues; Carboplatin and Iproplatin,Cyclophosphamide, Daunorubicin, Doxofluoridine, Doxorubicin, Etoposide,Epirubicin, 5-Flurouracil, Gemzar, Ifosfamide, Melphalan, Methotrexate,Mithramycin, Mitomycin C, Mitoxanthrone, Streptozotocin, Taxol andTaxotere, Vincristine, Vinblastine, Vindesine, Vinorelbine, Topotecanand CPT-11.

[0026] “Therapeutic” as used herein, includes treatment and/orprophylaxis. When used, therapeutic refers to humans, as well as, otheranimals.

[0027] “Pharmaceutically or therapeutically effective dose or amount”refers to a dosage level sufficient to induce a desired biologicalresult. That result may be the delivery of a pharmaceutical agent,alleviation of the signs, symptoms or causes of a disease or any otherdesirous alteration of a biological system.

[0028] A “host” is a living subject, human or animal, into which a drugor cytotoxic agent is administered.

[0029] Note, that throughout this application various citations areprovided. Each citation is specifically incorporated herein in itsentirety by reference.

[0030] The present invention includes a method for treating solid tumorscomprising administering to a host a therapeutically effective dose of acomposition comprising a PDGF aptamer and a cytotoxic agent. In apreferred embodiment the PDGF aptamer is identified using the SELEXmethodology. The SELEX process is described in U.S. patent applicationSer. No. 07/536,428, entitled “Systematic Evolution of Ligands byExponential Enrichment,” now abandoned, U.S. Pat. No. 5,475,096,entitled “Nucleic Acid Ligands,” and U.S. Pat. No. 5,270,163 (see alsoWO 91/19813), entitled “Methods for Identifying Nucleic Acid Ligands.”These applications, each specifically incorporated herein by reference,are collectively called the SELEX Patent Applications.

[0031] The SELEX process provides a class of products that are nucleicacid molecules, each having a unique sequence, and each of which has theproperty of binding specifically to a desired target compound ormolecule. Target molecules are preferably proteins, but can also includeamong others carbohydrates, peptidoglycans and a variety of smallmolecules. SELEX methodology can also be used to target biologicalstructures, such as cell surfaces or viruses, through specificinteraction with a molecule that is an integral part of that biologicalstructure.

[0032] In its most basic form, the SELEX process may be defined by thefollowing series of steps.

[0033] 1. A candidate mixture of nucleic acids of differing sequence isprepared. The candidate mixture generally includes regions of fixedsequences (i.e., each of the members of the candidate mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either: (a) to assistin the amplification steps described below; (b) to mimic a sequenceknown to bind to the target; or (c) to enhance the concentration of agiven structural arrangement of the nucleic acids in the candidatemixture. The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one in four) or onlypartially randomized (e.g., the probability of finding a base at anylocation can be selected at any level between 0 and 100 percent).

[0034] 2. The candidate mixture is contacted with the selected targetunder conditions favorable for binding between the target and members ofthe candidate mixture. Under these circumstances, the interactionbetween the target and the nucleic acids of the candidate mixture can beconsidered as forming nucleic acid-target pairs between the target andthose nucleic acids having the strongest affinity for the target.

[0035] 3. The nucleic acids with the highest affinity for the target arepartitioned from those nucleic acids with lesser affinity to the target.Because only an extremely small number of sequences (and possibly onlyone molecule of nucleic acid) corresponding to the highest affinitynucleic acids exist in the candidate mixture, it is generally desirableto set the partitioning criteria so that a significant amount of thenucleic acids in the candidate mixture (approximately 5-50%) areretained during partitioning.

[0036] 4. Those nucleic acids selected during partitioning as having therelatively higher affinity for the target are then amplified to create anew candidate mixture that is enriched in nucleic acids having arelatively higher affinity for the target.

[0037] 5. By repeating the partitioning and amplifying steps above, thenewly formed candidate mixture contains fewer and fewer uniquesequences, and the average degree of affinity of the nucleic acids tothe target will generally increase. Taken to its extreme, the SELEXprocess will yield a candidate mixture containing one or a small numberof unique nucleic acids representing those nucleic acids from theoriginal candidate mixture having the highest affinity to the targetmolecule.

[0038] The basic SELEX method has been modified to achieve a number ofspecific objectives. For example, U.S. patent application Ser. No.07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat. No.5,707,796, both entitled “Method for Selecting Nucleic Acids on theBasis of Structure,” describe the use of the SELEX process inconjunction with gel electrophoresis to select nucleic acid moleculeswith specific structural characteristics, such as bent DNA. U.S. patentapplication Ser. No. 08/123,935, filed Sep. 17, 1993, entitled“Photoselection of Nucleic Acid Ligands,” now abandoned, U.S. Pat. No.5,763,177 and U.S. Pat. No. 6,001,577, both entitled “SystematicEvolution of Ligands by Exponential Enrichment: Photoselection ofNucleic Acid Ligands and Solution SELEX,” all describe a SELEX basedmethod for selecting nucleic acid ligands containing photoreactivegroups capable of binding and/or photocrosslinking to and/orphotoinactivating a target molecule. U.S. Pat. No. 5,580,737, entitled“High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine,” describes a method for identifying highlyspecific nucleic acid ligands able to discriminate between closelyrelated molecules, termed Counter-SELEX. U.S. Pat. No. 5,567,588,entitled “Systematic Evolution of Ligands by Exponential Enrichment:Solution SELEX,” describes a SELEX-based method which achieves highlyefficient partitioning between oligonucleotides having high and lowaffinity for a target molecule. U.S. Pat. No. 5,496,938, entitled“Nucleic Acid Ligands to HIV-RT and HIV-1 Rev,” describes methods forobtaining improved nucleic acid ligands after SELEX has been performed.U.S. Pat. No. 5,705,337, entitled “Systematic Evolution of Ligands byExponential Enrichment: Chemi-SELEX,” describes methods for covalentlylinking a ligand to its target.

[0039] The SELEX method encompasses the identification of high-affinitynucleic acid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified nucleic acid ligands containingmodified nucleotides are described in U.S. Pat. No. 5,660,985, entitled“High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,”that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat.No. 5,637,459, supra, describes highly specific nucleic acid ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH₂),2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent applicationSer. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method ofPreparation of Known and Novel 2′ Modified Nucleosides by IntramolecularNucleophilic Displacement,” describes oligonucleotides containingvarious 2′-modified pyrimidines.

[0040] The SELEX method encompasses combining selected oligonucleotideswith other selected oligonucleotides and non-oligonucleotide functionalunits as described in U.S. Pat. No. 5,637,459, entitled “SystematicEvolution of Ligands by Exponential Enrichment: Chimeric SELEX,” andU.S. Pat. No. 5,683,867, entitled “Systematic Evolution of Ligands byExponential Enrichment: Blended SELEX,” respectively. These applicationsallow the combination of the broad array of shapes and other properties,and the efficient amplification and replication properties, ofoligonucleotides with the desirable properties of other molecules.

[0041] In U.S. Pat. No. 5,496,938, methods are described for obtainingimproved nucleic acid ligands after the SELEX process has beenperformed. This patent, entitled “Nucleic Acid Ligands to HIV-RT andHIV-1 Rev,” is specifically incorporated herein by reference.

[0042] One potential problem encountered in the diagnostic use ofnucleic acids is that oligonucleotides in their phosphodiester form maybe quickly degraded in body fluids by intracellular and extracellularenzymes, such as endonucleases and exonucleases, before the desiredeffect is manifest. Certain chemical modifications of the nucleic acidligand can be made to increase the in vivo stability of the nucleic acidligand or to enhance or to mediate the delivery of the nucleic acidligand. See, e.g., U.S. patent application Ser. No. 08/117,991, filedSep. 8, 1993, now abandoned and U.S. Pat. No. 5,660,985, both entitled“High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,”and U.S. patent application Ser. No. 09/362,578, filed Jul. 28, 1999,entitled “Transcription-free SELEX,” each of which is specificallyincorporated herein by reference in its entirety. Modifications of thenucleic acid ligands contemplated in this invention include, but are notlimited to, those which provide other chemical groups that incorporateadditional charge, polarizability, hydrophobicity, hydrogen bonding,electrostatic interaction, and fluxionality to the nucleic acid ligandbases or to the nucleic acid ligand as a whole. Such modificationsinclude, but are not limited to, 2′-position sugar modifications,5-position pyrimidine modifications, 8-position purine modifications,modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo or 5-iodo-uracil; backbone modifications,phosphorothioate or alkyl phosphate modifications, methylations, unusualbase-pairing combinations such as the isobases, isocytidine andisoguanidine and the like. Modifications can also include 3′ and 5′modifications such as capping. In preferred embodiments of the instantinvention, the nucleic acid ligands are RNA molecules that are 2′-fluoro(2′-F) modified on the sugar moiety of pyrimidine residues.

[0043] The modifications can be pre- or post-SELEX processmodifications. Pre-SELEX process modifications yield nucleic acidligands with both specificity for their SELEX target and improved invivo stability. Post-SELEX process modifications made to 2′-OH nucleicacid ligands can result in improved in vivo stability without adverselyaffecting the binding capacity of the nucleic acid ligand.

[0044] Other modifications are known to one of ordinary skill in theart. Such modifications may be made post-SELEX process (modification ofpreviously identified unmodified ligands) or by incorporation into theSELEX process.

[0045] The nucleic acid ligands to PDGF of the invention are preparedthrough the SELEX methodology that is outlined above and thoroughlyenabled in the SELEX applications incorporated herein by reference intheir entirety.

[0046] As noted above, the cytotoxic agent can be any substance used inthe prevention, diagnosis, alleviation, treatment or cure of disease.More specifically, the cytotoxic agent can be selected from anysystemically administrated agent including, but not limited to,Bleomycin, Cisplatin, and Pt analogues; Carboplatin and Iproplatin,Cyclophosphamide, Daunorubicin, Doxofluoridine, Doxorubicin, Etoposide,Epirubicin, 5-Flurouracil, Gemzar, Ifosfamide, Melphalan, Methotrexate,Mithramycin, Mitomycin C, Mitoxanthrone, Streptozotocin, Taxol andTaxotere, Vincristine, Vinblastine, Vindesine, Vinorelbine, Topotecanand CPT-11.

[0047] Various delivery systems are known in the art and can be used toadminister the therapeutic composition comprising the PDGF aptamer andcytotoxic agent of the invention, e.g., aqueous solution, encapsulationin liposomes, microparticles, and microcapsules.

[0048] Therapeutic compositions of the invention may be administeredparenterally by injection, although other effective administrationforms, such as intraarticular injection, inhalant mists, orally activeformulations, transdermal iontophoresis or suppositories are alsoenvisioned. One preferred carrier is physiological saline solution, butit is contemplated that other pharmaceutically acceptable carriers mayalso be used. In one preferred embodiment, it is envisioned that thecarrier and the nucleic acid ligand constitute aphysiologically-compatible, slow release formulation. The primarysolvent in such a carrier may be either aqueous or non-aqueous innature. In addition, the carrier may contain otherpharmacologically-acceptable excipients for modifying or maintaining thepH, osmolarity, viscosity, clarity, color, sterility, stability, rate ofdissolution, or odor of the formulation. Similarly, the carrier maycontain still other pharmacologically-acceptable excipients formodifying or maintaining the stability, rate of dissolution, release orabsorption of the ligand. Such excipients are those substances usuallyand customarily employed to formulate dosages for parentaladministration in either unit dose or multi-dose form.

[0049] Once the therapeutic composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or dehydrated or lyophilized powder. Such formulations may be storedeither in a ready to use form or requiring reconstitution immediatelyprior to administration. The manner of administering formulationscontaining the compositions for systemic delivery may be viasubcutaneous, intramuscular, intravenous, intranasal or vaginal orrectal suppository.

[0050] The amount of the composition which will be effective in thetreatment of a particular disorder or condition will depend on thenature of the disorder of condition, which can be determined by standardclinical techniques. In addition, in vitro or in vivo assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness or advancement of thedisease or condition, and should be decided according to thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curved derived from in vitro or animalmodel test systems. For example, an effective amount of the compositionof the invention is readily determined by administering graded doses ofthe composition of the invention and observing the desired effect.

[0051] The following examples are provided to explain and illustrate thepresent invention and are not intended to be limiting of the invention.

[0052] The PDGF-B aptamer used in the present study ((SEQ ID NO:1), FIG.1A) was produced by the SELEX method. (Tuerk and Gold (1990) Science249:505-510, which is incorporated herein by reference in its entirety).The modified DNA aptamer, linked to 40 kDa polyethylene glycol, has ahigh affinity for PDGF-B with a Kd of˜0.1 nM. (Green et al. (1996)Biochemistry 35:14413-14424; Floege et al. (1999) Am. J. Pathol.154:169-179; U.S. Pat. No. 6,229,002, issued May 8,2001, ((SEQ IDNO:146), FIG. 9A), each of which is incorporated herein by reference inits entirety). A sequence-scrambled analog of the PDGF-B aptamer wasused as a control. ((SEQ ID NO:2), FIG. 1B). This oligonucleotide has aKd for PDGF-BB in the micromolar range. (Floege et al. (1999) Am. J.Pathol. 154:169-179; U.S. Pat. No. 6,229,002, issued May 8, 2001, ((SEQID NO:147), FIG. 9B)).

[0053] Example 1 describes the method used to treat PROb tumor-bearingrats with the PDGF-B specific aptamer. As can be seen in FIG. 2treatment of PROb tumor-bearing rats with the PDGF-B specific aptamerresulted in a decrease in tumor IFP when compared to rats treated withthe control aptamer. The mean IFP in control aptamer-treated tumors was14.6±1.2 mm Hg (±S.E.M.) and 9.7±1.6 mm Hg (±S.E.M.) in tumors treatedwith the PDGF-B specific aptamer. The method used to determine IFP isdescribed in Example 2.

[0054] PROb tumors were analyzed with regard to morphology, as well asdistribution of PDGF-AB/BB and PDGF β-receptors as described in Example3. The tumors displayed a heterogeneous morphology. At the tumorperiphery, tumor cells were arranged in glandular structures, whereasmore centrally, tumor cells were less abundant and less well organized(FIG. 3A). The central part was basically acellular (FIG. 3A).Expression of PDGF-AB/BB in PROb tumors was found in blood vessels andpossibly in extravascular stromal cells surrounding tumor glands (FIG.3B). In the central part of the tumors, few if any tumor cells werepresent, but strongly PDGF-AB/BB positive cells were seen (FIG. 3C). Inno part of the tumors could PDGF-AB/BB positive tumor cells be clearlydiscerned. PDGF β-receptors were found in vascular cells of largervessels, and in unidentified, possibly microvascular, cells in thestroma (FIG. 3D). The absence of PDGF-AB/BB and β-receptor expression bycarcinoma cells in PROb tumors, is in agreement with the characteristicsof cultured PROb cells (data not shown).

[0055] Prompted by these findings, the effects of treatment with PDGFaptamer (SEQ ID NO:1) were tested on the KAT-4 tumor model (Examples 4and 5). Both of these tumor models showed PDGF receptor expression intumor stroma but not on tumor cells. As can be seen in FIGS. 4 and 5treatment with PDGF aptamers lowers IFP in KAT-4 tumors and increasesthe uptake of Taxol.

EXAMPLES

[0056] Materials and Methods

[0057] Tissue culture. Cells were cultured under standard conditions andall tissue culture media were supplemented with 10% fetal bovine serum(FBS) and antibiotics, unless otherwise stated. PAE cells weremaintained in F12 culture medium (Sigma). PROb cells were kept inDulbecco's Modified Eagle's Medium (Sigma).

[0058] PDGF/PDGF receptor inhibitors. The PDGF-B aptamer used in thepresent study was produced by the Systematic Evolution of Ligands byExponential Enrichment (SELEX) method. (Tuerk and Gold (1990) Science249:505-510, which is incorporated herein by reference in its entirety).The modified DNA aptamer, linked to 40 kDa polyethylene glycol, has ahigh affinity for PDGF-B with a Kd of˜0.1 nM. (Green et al. (1996)Biochemistry 35:14413-14424; Floege et al. (1999) Am. J. Pathol.154:169-179, each of which is incorporated herein by reference in itsentirety). The aptamer shows biphasic clearance in rats following ivinjection, approximately 47% is cleared with a half-life of 32 minutes,while the remainder is cleared with a half-life of 135 minutes. (Floegeet al. (1999) Am. J. Pathol. 154:169-179). As a control, asequence-scrambled analog of the PDGF-B aptamer was used. Thisoligonucleotide has a Kd for PDGF-BB in the micromolar range. (Floege etal. (1999) Am. J. Pathol. 154:169-179).

Example 1 Tumor Establishment and Treatment of PROb Tumors with PDGFInhibitors

[0059] Subcutaneously growing PROb tumors (Martin et al. (1996) Int. J.Cancer 65:796-804) were established in BDIX rats by injection of 5×10⁶tumor cells in 50 μL of PBS in the flank. The rats were kept underpathogen-free conditions and were fed ad libitum. They were monitoredregularly for tumor growth and experiments were performed 8-12 weeksafter tumor cell implantation on rats bearing tumors ranging in sizebetween 0.6 cm³ and 7.6 cm³. The PDGF-B specific aptamer, and a controlaptamer (see above), were given as i.p. injections in 2 mL PBS twicedaily for 4 consecutive days at a dose of 7 mg×kg⁻¹×day⁻¹. All animalexperiments described in the present report were approved by the EthicalCommittee for Animal Experiments in Uppsala, Sweden.

Example 2 Measurement of Tumor IFP

[0060] Tumor IFP was measured using the wick-in-needle technique (Wiiget al. (1982) Scan. J. Clin. Lab. Invest. 42:159-164). Briefly, ratswere anaesthetized using isofluran in a mixture of O₂ and air. Astandard 23-gauge needle filled with nylon-floss and saline,supplemented with 50 IE/mL of heparin was inserted into the center ofthe tumor and connected to a pressure transducer. This makeup enablescontinuous and stable recordings of fluid pressure. Fluid communicationbetween the needle and the transducer was confirmed by compression anddecompression of the tubing during each measurement. Tumor IFP wasmeasured once before treatment with PDGF aptamers, and again 1-2 hoursafter the last administration of aptamers or vehicle alone. The changein tumor IFP was calculated for each tumor. After the second IFPmeasurement the rats were sacrificed and the tumors were excised andsnap frozen in liquid nitrogen for further analyses.

Example 3 Immunohistochemistry

[0061] For routine morphology, paraffin-embedded 4 μm sections werestained with van Gieson staining. Immunohistochemistry was performed on6 μm cryosections from PROb tumors. Sections were fixed in acetone andblocked with 0.3% hydrogen peroxide in methanol for 15 minutes, rinsedand further incubated in a solution containing 20% human normal serum ina buffer containing 2% rat serum, 3% bovine serum albumin, 0.01% NP40 inPBS (RM buffer) for 5 hours at 4° C. Primary antibodies dissolved in RMbuffer were added, either 4 μg/mL affinity-purified rabbit anti-PDGFβ-receptor IgG (Claesson-Welsh et al. (1988) Mol. Cell Biol.8:3476-3486) for 5 hours at 4° C., or overnight at 4° C. with 1.3 μg/mLof the monoclonal mouse anti-PDGF-AB/BB IgG (PGF 007, MochidaPharmaceutical Company, Tokyo, Japan). Sections were rinsed in PBS with0.01% NP40. Bound IgG was detected with biotinylated goat anti-rabbit orbiotinylated rabbit anti-mouse antibodies, respectively. Sections weredeveloped with a Vectastain ABC elite kit (Vector, Burlingame, Calif.)using amino-ethyl-carbazole as a chromophore. Sections werecounter-stained with Mayer's hematoxylin for 30 seconds.

Example 4 Treatment of KAT-4 tumors with PDGF Inhibitors

[0062] SCID-mice bearing subcutaneous KAT-4 tumors (human, anaplasticthyroid carcinoma) were pre-treated for 4 consecutive days with 12mg×kg⁻¹×day⁻¹ SELEX aptamers (i.p. injections, three times daily). Tumorinterstitial fluid pressure was measured using the wick-in-needletechnique. The results are set forth in FIG. 4. * p<0.05, Student'st-test.

Example 5 Treatment of KAT-4 tumors with a PDGF Inhibitor in Combinationwith the Cytotoxic Agent Taxol

[0063] SCID-mice bearing subcutaneous KAT-4 tumors (human, anaplasticthyroid carcinoma) were pre-treated for 4 consecutive days with 12mg×kg⁻¹×day⁻¹ SELEX aptamers (i.p. injections, three times daily).[³H]Taxol was injected s.c at a site distant from the tumor and in a mixof 5 mg×kg⁻¹ unlabelled Taxol. Eight or 24 hours following injection ofradiolabelled drug, blood was sampled, animals were sacrificed andtumors and 4 other tissues were excised. Subsequently, tissues wereweighed and homogenized in a RIPA lysis buffer and the amountradioactivity in each sample was determined in a scintillation counter.Tumor uptake of Taxol was expressed as cpm/g tumor tissue divided bycpm/ml blood. The results are set forth in FIG. 5. * p<0.05, Student'st-test.

[0064] Statistical analysis. Statistical analysis was performed usingthe paired or unpaired two-sided Student's t-test. A p-value<0.05 wasconsidered statistically significant.

What is claimed is:
 1. A method for treating tumors comprisingadministering to a host a therapeutically effective dose of acomposition comprising a platelet-derived growth factor (PDGF) aptamerand a cytotoxic agent.
 2. The method of claim 1 wherein said PDGFaptamer is identified according to a method comprising: a) preparing acandidate mixture of nucleic acids; b) contacting the candidate mixtureof nucleic acids with PDGF, wherein nucleic acids having an increasedaffinity to PDGF relative to the candidate mixture may be partitionedfrom the remainder of the candidate mixture; c) partitioning theincreased affinity nucleic acids from the remainder of the candidatemixture; and d) amplifying the increased affinity nucleic acids to yielda mixture of nucleic acids enriched for nucleic acids with relativelyhigher affinity and specificity for binding to PDGF, whereby a nucleicacid ligand of PDGF may be identified.
 3. The method of claim 1 whereinsaid PDGF aptamer is SEQ ID NO:1.
 4. The method of claim 1 wherein saidcytotoxic agent is selected from the group consisting of Bleomycin,Cisplatin, and Pt analogues; Carboplatin and Iproplatin,Cyclophosphamide, Daunorubicin, Doxofluoridine, Doxorubicin, Etoposide,Epirubicin, 5-Flurouracil, Gemzar, Ifosfamide, Melphalan, Methotrexate,Mithramycin, Mitomycin C, Mitoxanthrone, Streptozotocin, Taxol andTaxotere, Vincristine, Vinblastine, Vindesine, Vinorelbine, Topotecanand CPT-11.
 5. A method for reducing the interstitial fluid pressure(IFP) of a tumor comprising administering a PDGF aptamer.
 6. The methodof claim 5 wherein said PDGF aptamer is identified according to a methodcomprising: a) preparing a candidate mixture of nucleic acids; b)contacting the candidate mixture of nucleic acids with PDGF, whereinnucleic acids having an increased affinity to PDGF relative to thecandidate mixture may be partitioned from the remainder of the candidatemixture; c) partitioning the increased affinity nucleic acids from theremainder of the candidate mixture; and d) amplifying the increasedaffinity nucleic acids to yield a mixture of nucleic acids enriched fornucleic acids with relatively higher affinity and specificity forbinding to PDGF, whereby a nucleic acid ligand of PDGF may beidentified.
 7. The method of claim 5 wherein said PDGF aptamer is SEQ IDNO:1.
 8. A method for increasing the uptake of cytotoxic agents into atumor comprising administering to a host a composition comprising a PDGFaptamer and a cytotoxic agent.
 9. The method of claim 8 wherein saidPDGF aptamer is identified according to a method comprising: a)preparing a candidate mixture of nucleic acids; b) contacting thecandidate mixture of nucleic acids with PDGF, wherein nucleic acidshaving an increased affinity to PDGF relative to the candidate mixturemay be partitioned from the remainder of the candidate mixture; c)partitioning the increased affinity nucleic acids from the remainder ofthe candidate mixture; and d) amplifying the increased affinity nucleicacids to yield a mixture of nucleic acids enriched for nucleic acidswith relatively higher affinity and specificity for binding to PDGF,whereby a nucleic acid ligand of PDGF may be identified.
 10. The methodof claim 8 wherein said PDGF aptamer is SEQ ID NO:1.
 11. The method ofclaim 8 wherein said cytotoxic agent is selected from the groupconsisting of Bleomycin, Cisplatin, and Pt analogues; Carboplatin andIproplatin, Cyclophosphamide, Daunorubicin, Doxofluoridine, Doxorubicin,Etoposide, Epirubicin, 5-Flurouracil, Gemzar, Ifosfamide, Melphalan,Methotrexate, Mithramycin, Mitomycin C, Mitoxanthrone, Streptozotocin,Taxol and Taxotere, Vincristine, Vinblastine, Vindesine, Vinorelbine,Topotecan and CPT-11.